The micronucleus test: A method used in mutagenicity testing

(1)

The

Micronucleus

Test:

a

Method Used

in Mutagenicity

Testing

Jeanne

Adiwinata

Pawitan

Abstrak

Mikronukleus adalah suatu bangunan bulat di dalan sitoplasnn, yang nrcngandu,tg ,,tassa kronatin (DNA). Mikrottukleus

tinbul

bila suatu populasi sel yang sedang nentbelah utengalatni patah kronosotn yang disebabknn oleh klastogen (sesuatu yang nrcnyebabknn patah krontoson), atau ntengalani hilangnT,a kronosour karena disfungsi spindel nitotilc

IIji

nikronukleus adalah cara cepat wûuk ntenguji nutagenisitas berbagai bahan.

Uji

itti juga dapat digunakan untuk nenilai kerusakan sitogenetikyang disebabkan paparan bahan genotoksik, dan telah terbukti satna sensitifnya dengan petneriksaan sitogenetik Makalah ini tnetnbicarakan tentang pene,,tuan mikronukleus, penyebab terbetûulaq,a nûkronukleus dan berbagai cspek uji nikronukleus. Mikronukleus dapat dianalisa pada berbagai sel, secarainvivo pada berbagai individu (nnnusia, hewan, dan tatranan), atau invitro padabiakan seI. Selyang paling sering diperiksa pada uji nùkronukleus itt vivo adalah eritrosit dan dan pada uji mikronukleus in vitro adabh linfosit. Penggunaan pewarnafluoresens pada uji nikronuHeus nentberi hasil yang lebih dapat dipercaya, tetapi penggunaannya terbatos, karena waktufluoresensinya terbatas.

Di nnsa datang, hal ini dapat _{diatasi dengan cara penghitutlgan yan7 nenggunakan Flow-sitoneter. Pewarnaan nikronukleus yang} berdasarlcan _{pewarna Feulgen ntenberi ganbaran )ta,lg kura,lg kontras, sehingga nenl'ulitkan pada penghitungan secara unnual,} nantun hasilnya daltat dipercaya. Dinnsa datang hal ini dapat diatasi dengan cara penghitungan oto,ilatik

Abstract

Micronucleus is

a

c7,16111ss1ûc

rontd

bodl' v'hich contains

chronatin

nass (DNA). Micronuclei arise when replicating cell populatiotts are subiected to chrontosotttal breaknge by clastogens (agents causing chrontosonal breakrtge) or to chronrosotne loss by ntitotic spindle d1'sfunctiotr. The nicronu.cleus test is a rapid nethod to test the ilrutagenicit5,of various agents. This test can also be used to 4.sses's the cytogenetic dantage caused by e\posures to genotoxic agents, and is proven to be as sensitive as the laborious cl,togenetic anal5'sis. This paper discuss the discovery ofnticronuclei, the cause ofnicronuclei _fornationand the various aspects ofthe tnicronu.cleus test. Micronuclei can be analS,Tsi in various

celk,

itr vivo usitrg various individuals (hunnn, aninak, and plants), or

in vitro

using cultured cells. The nostfrequ.ently screened celLs in in vivo micronucleus test are the erlthrocytes, and in in vitro nticronu.cleu.s test are the lynryhocytes. The use offluorescent stain in uicronucleus test gives a,ilore reliable resuh, but the use is lituited, due to the litnited

fluorescent tinte. In the _future,this can be overcoue using Flow-cylonetric enwneration. The Feulgen based staining of nùcronuclei gives

a less

well pronowtced inage, thus

dificult

to score nnnually, though the resuh is reliable. In thefuture this can be overconte

usi ng auto,nated enwneratiott ilrcthod.

Keywords: _{Polychronatic erylhrocy1ss, binucleated lynphocytes, Gienna}_stain,_{Feulgen-based}_slain,_{acridin orange}_stain, Hoechst-pyronin stain, irnage analysis, autonated detection, _flow

q'ronetrt-The

micronucleus

is a

small distinct

extranuclear

body

in the

cytoplasm

of

a

cell. It is

spherical

in

shape

with

a diameter

smaller than

1/3

of

the diameter

of

the

main

nucleus

and has a

similar

staining

quality

as the

main

nucleus.

The number

of

micronuclei

varies

between

one

to three

per

cell

(most

cells carry

only

one

micronucleus, some carry 2-3, and cells carrying

4

m

e extremely

rare).

w

nucleus,l

and

ii

co

m

Micronucleus pers

able

period of time,

and

it

still

persjsts after one or two

cell divisions.l

The term

"micronucleus

test"

was

originally

coined

for detecting chrontosome breaks

induçcd.by

(2)

cytogenetic

damage caused

by

exposures to

genotoxic

agents, and is

claimed

to be as

reliable

as the

laborious

cytogenetic

analysis.

This

paper discuss the

discovery

of

micronuclei,

the

cause

of

micronuclei formation

and

the

various

aspects

of

the micronucleus test,

including

the cells

screened

for in vivo or in vitro

micronucleus lest,

the

animals

used

in

the test,

and the

micronuclei

staining

and

enumeration

methods.

THE DISCOVERY OF MICRONUCLEI

ln

7946, Catcheside

et

al.

described

additional

nuclei

(now most

likely

called

micronuclei) which

arrised in

cells

subjected

to radiation.

Five

years

later

Thoday

observed

the

presence

of

more than one

nucleus

in

some

of

the

proliferating

cells

in

the

root

lips

of

Vicia

faba,

some time after irradiation.

These additional

nuclei

were smaller

in

size than the main

one, and he

called

these structures micronuclei

or

fragment

, . cill

nuclel.

TIIE

CAUSE OF MICRONUCLEI FORMATION

Micronuclei

arise when

replicating

cell

populations are

subjected

to

chromosomal

breakage

by clastogens

(agents causing chromosomal breakage)

or

to

chromosome

loss

by

mitotic

spindle dysfunction./

Chromosome

breakage

will

give rise to acentric

frag-ments at mitosis,

These

fragments,

due

to

the lack

of

kinetochore

will

be excluded

from

the

two

new

daughter nuclei

at

telophase,

and

will

appear

as

micronuclei in one

or both

of the

daughter cells.

There-fore,

it

was supposed

that micronuclei

enclosed

acentric

chromosome

fragments,

or,

in

case

of

ex-posure to an agent cau-sing

mitotic

spindle dysfunction,

they

might contain whole

chromosomes, that

had not been

incorporated

in

the main

nuclei

at

cell division.l

However, later

it

is

believed that

micronuclei

can enclose

acentric

or centric

chromosome fragments or

whole

chromosomes.4

TIIE

CELLS SCREENED FOR

MICRONUCLEI

The micronucleus test

is now widely

used in

mutagenicity

testing, due to its

simplicity. Micronuclei

can be

induced

in vivo

or

in vitro.

Thus, the test can be

done

in

vivo

using men, animals such as

mice,2'6

chick,g

fish,13

newt

larvae,s

etc. and

plants,14

or

in

vitro

using cultured

cells.a'8'10'15

The most frequently

screened

cells

in in

vivo

micronucleus test are

the

erythrocytes,

and

in in vitro

micronucleus

lest. are the

lymphocytes.

Screening

the Erythrocytes

The

chromosome

breaks induced

in

erythrocytes

before maturation,

condense themselves into

micronuclei (in

erythrocytes, they

are

also called

the

Howell

Jolly

bodies).

While

the main nucleus

is

ejected

during maturation,

the

micronuclei

persist, and

can be detected easily.

In

mice, micronucleated

erythrocytes

are not selectively removed

from

the

peripheral

blood. However,

the spleen

of

human

and

most animals,

removes

micronucleated

erythrocytes

from

the

circulation.

Therefore,

the specimen used

for

scoring micronucleated cells is

usually

the

adult

bone

marrow.

The bone

marrow contains a variety of blood

cells

and

their

precursors;

in

bone

marrow,

the

micronuclei are usually

scored

in

polychromatic

erythrocytes.

However, some investigators

used

the

peripheral

blood

from

splenectomized

^.n,7

and

some

cardiac blood

erythro-cytes

of

newt

larv

les wahl).s

The reactive forms

of

chromosome-damaging

chemicals

are

often metabolites formed

within

tissues

of

exposed

individuals. Adult

bone

marrow

has

limited

activation capacity, and short

lived

mutagens/car-cinogens

formed

in other tissues may not

survive

in the

circulation,

so

false-negative results

can be obtained. Late

in

gestation,

blood cells develop

in

the

fetal

liver,

alongside

hepatocytes

which

metabolize

premutagens to active

forms. A

study,

exposing

l5-

16 days pregnant Swiss

albino

mice

to

a premutagen and

screening

the

polychromatic erythrocytes, proved that the

highest

percentage

of

the micronucleated erythrocytes

was

found in fetal liver, followed by fetal blood,

and

the

lowest

was

in

maternal bone marrow.o Therefore,

for

premutagens

that

need

activation,

screening

in

fetal

liver

or fetal blood is preferable.

The fetal blood

contains

the

erythrocytes, other

blood cells

and

their

precursors.

Among

the

erythro-cytes,

a

large number

of very young

(polychromatic)

and

young

(stippled)

erytrhrocytes

are present.

In

the

micronucleus

test, screening the

fetal erythrocytes

can be done on the

polychromatic erythrocytes

only,6 or on

all

erythrocytes.T

The highest incidence

of polychromatic

erythro-cytes

containing micronuclei in fetal liver was

12-18

hours

after

the

effective

exposure

to

the

premutagen, and

in both

maternal bone

marrow

and

fetal blood

was 30 hours

after

the

effective

"r,porrr.".6

(3)

Table

l.

The spontaneous _{insidence of micronucleated cells}

in

_{various individuals} Specimen

origin

Specimen

Type of cell

micronucleated

Number

of

Number _cells

of

cell

screened

Staining

Mouse*

Human

f

(sp ITP)

Human

f

(sp trauma)

Human

f

(healthy)

fetal liver

fetal blood

peripheral blood

polychromatic erythrocyte

polychromatic erytfuocyte

total erythrocyte

cytokinesis blocked lymphocye

1.610.3

4.010.8

2.4

t

0.9

2.1

r

0.3

2.4

!

O.7

4.4

!

2.6 adult

bone manow

1000

500

May Grun-wald Giemsa

May Grun-Giemsa

Wright

May Grun-wald Giemsa

*

_{From Cole}_{et a1.,6}

t

_{from Schlegel}_et_a1.,7

I

_from_{Fenech and}_Morley,lo_sp_ITP

= _{splenectomized for idiopathic} thrombocyto-penic purpura, sp _{trauma = splenectomized for}_trauma.

Screening

the lymphocytes

In human, the difficulty

of

obtaining

bne

marrow

samples makes the

in vivo

method

impossible to

be used

in

large scale

investigations.

Since the

formation

of

micronuclei is a

general phenomenon

in

dividing

damaged

cells,

it

is also possible

to study them in

cultured

lymphocytes.

The

first

attempt

to

score

micronuclei

in

lymphocytes

was done after the

cytoplasm

had been destroyed

by hypotonic

treatment.

Countryman and Heddle (1976) and Heddle

et

al.

(1978) found that

exposrues

to

X-ray radiation

and

mitomycin

C

in

vitro

lead to a dose-related increase

of

micronuclei in

cultured lymphocytes.

Scoring micronuclei

in

hypotonically

treated

preparations, displays certain

disadvantages, because the

cytoplasm

is not

_{preserved and}

the

nuclei

tend to

clump, making

the

evaluation

difficult.l

Moreover,

it

is

often

difficult

to differentiate real

micronuclei from

nuclear/cellular debris from other cells,

because the

cytoplasm

of

the

cell is lacking.

Cells

with

segmenta-tion

or

fragmentation

of

the nucleus can easily

_be

regarded

as lymphocytes

with micronuclei.

As

the

cytoplasm

of

the

cells is

destroyed,

micronuclei

may

also

be

torn from

the neighbourhood

of

the

original

nucleus.s

Therefore,

modiiications

were presented.

Is-kan

ld

hypoto

ofa

ogicalsal

and

chloride

treat

the

cultured

lymphocytes. The osmotic

_pressure

of this mixture is

285

milliosmol.l5

The

modification

tes. This

Hogstedt

duals

ex-posed

in

vivo,

or lymphocytes

exposed

in

vitro,

to

X-rays,

and eliminated

the hypotonic

treatment.8

Using this method,

the

cytoplasm is

also preserved.

The fo

division to

methods are [image:3.595.47.554.100.385.2]

(4)

appearance.

This method is simple

to

perform,

as a

large number

of

these

cells

could be accumulated by

adding

3.0

Fe/ml cytochalasin B

at 44 h and

scoring at

72h.to

According to

Fenech

et

al. (1984),

for

lympho-cytes

irradiated

in vitro there

is a linear

relationship

between dose

radiation and the number

of

induced

micronuclei; the

in

vivo

micronucleus frequency in

cytokinesis-blocked lymphocytes

from

normal

in-dividuals

is

shown

in

fuUt"

r.tô

Screening

other

cells

in the in vivo micronucleus

test

used

cells

from

Screening

the hepatocytes

The hepatocytes are most exposed to mutagens, due to the

detoxification function

of

the

liver.

Especially

for

premutagens

which

need

activation, scoring

the

micronucleus

in

hepatocytes

should

be

very sensitive.

In

mice, the micronucleated

cells

increase

in

correla-tion with

the

increasing

age (Table2).16

Table

2.

The proportion of micronucleated hepatocytes

in

nrice of different ages*

Age (months) Micronucleated hepatocytes (%o)

5.9 8.9 59.3 I17.0

* _Resumed_{from Uryvaeva}_{and Delone.16}

Screening

the

exfoliated cells in

spututtt

Micronuclei reflect

DNA

damage

in

exfoliated

cells

and

may thuç provide

a

marker

of

early-stage

car-cinogenesis." Micronuclei

can be

found in

exfoliated

cells

in

sputum of heavy smokers, and they can be used to evaluate substances that may reduce lung cancer

risk

in

man.l7

Scre ening the

fibroblast

The cytokinesis-block method using cytochalasin B

was applied

to

the primary fibroblast culture.

The

fibroblast

were

obtained

from

Fischer

(F34Q

rats

that

had

been

treated

by

ethylnitrosourea

in

vivo

for

5

hours. The number

of micronuclei

reached a

maximum

on the

third

day.18

Screening

other

cells

in the in

vitro

micronucleus

test

In addition to

ly

e

usedsuchasthe

e

Mewo

(human

e

Chinese hamste

THE

MICRONUCLEI STAINING METHOD

The micronucleus

consists

of

the same

material

as the

main nucleus; consequently, the staining

methods

usually

used

to

stain the nuclei, chromosomes, or

DNA

should

be able

to stain

the

micronucleus.

There-fore,

several

staining

methods was

applied

to stain the

micronuclei

.z't 'zt 'zej's

The

methods

using

modifications

of

the

Romanowsky stain

In 1890,

Romanowsky observed that,

by mixing

old,

mold-covered solution of methylene blue

with

eosin, a

stain was

produced

which

stained the

blood cells very

well. A

purplish-red stain,

the

methylene

aztrre,

which

is an oxidation product of methylene blue, is

produced

when

moldy

methylene

blue is mixed with

eosin.

In

1891,

Malachowski

demonstrated

that

this

new

metachromatic

stain

could

be obtained

consistently by

alkalinization

of methylene

blue

and

the

subsequent

addifion

of eosin, thus

making

it

unnecessary

to wait

for

mold formation

to

occur. Following

these

dis-coveries, numerous modifications was made;

these

include the

stains

of

Wright

(1902)

and

Giemsa

(tgo2).21

The

Wright

and

Giemsa staining method

and

its

modifications (including the May

Grunwald-Giemsa

staining

method) are

often

used

to

stain

the

micronuclei. With

these

staining

methods, the nuclei

and

micronuclei

appear deep

purple, red

purple,

purplish blue

or magenta,

but the

RNA

appears the

same.

Staining the

fetal

blood using

these staining

methods,

will

stain the

RNA within

the very young and

young erythrocytes.

In

very young (polychromatic)

erythrocytes,

the

RNA

is stained

uniformly,

and

won't

give any problem. However,

the

RNA in

young

(stip-pled)

erythrocytes

(also called reticulocytes) gives

a

fine stippling

appearance.

These

stipples

tend

to

[image:4.595.53.287.462.590.2]

(5)

Inadiation MMS 25 pglml Inadiation MMS 25 pg/ml

37.7 + 36.5 44.7 + 23.9 23.5

+

2.1

22.7 + 10.5

gregate

and thus

ressemble

a

micronucleus.2

There-fore,

the

Romanowsky-based staining

methods is used

only

to screen the

polychromatic erythrocytes; scoring

in

the stippled erythrocytes is

unreliabte, due

to

the presence

of

numerous

RNA

aggregates.z

Staining the lymphocytes using

these staining

methods,

will

also stain

the dense lysosomes

that

ap-pear as

tiny

reddish.-p^urple

granules

called

the

azurophilic

granules.'""'"

Roughly

lO%

of

lym-phocytes contain

these

granules.'"

It

is difficult

to

distinguish the reddish purple azurophilic

granules

from

the

micronucleus,

so

scoring

in

the

lymphocytes

using

these

staining

methods

will

give a higher

fre-quency

of

micronuclei,

compared

to scoring using

a

DNA specific

(Feulgen) stain (Table

3).

Table

3. The

proportion

of

micronucleated cetls stained with Giemsa or Feulgen4ongo red, in binucleated lymphocSrtes, after treatment*

Treatment Staining method

tions,

RNA

fluorescès

red, and

DNA

(micronuclei)

green-yellow.7

The Hoechst 33258-pyronin

Y

stain

consists

of

Hoechst 33258

which

stains

only

DNA,

and

pyronin

y

which

stains

both

DNA

and

RNA.

Stained slides

are

examined

by

fluorecent microscopy. Excitation

of

pyronin

with

green

light

(546 nm)

causes

both RNA

and

DNA

to

fluoresce orange,

while

hoechst

stained

DNA

(but not

RNA)

fluoresces

brilliant

blue under

UV

(360

nm)

excitation.

The RNA-

negative

(mature)

erythrocytes are visualized using

dim

brightfield

(Koehler) illumination.'

The Hoechst

33342-thiazole

orange stain consists

of

thiazole orange that

is

used

for discrimination

be-tween polychromatic and normochromatic

erythro-cytes,

and Hoechst

33342

that

is

used

to

detect

the micronucleus.24

The

use

of

fluorescent

stains needs fluorescent

mycroscopy

to

examine

the slides.

In addition,

screen-ing

must be done

quickly,

due to the

limited

fluorescent

time.

Consequently,

the

use

of

fluorescent

stains

in

screening micronucleated

cells is limited,

though

the

result is more reliable.

The F e ul g e n-bas e d

staining

nt etho

d

The Feulgen-Congo red

(FCR)

staining method

com-bines

the

Feulgen

stain which

stains

only

DNA

and

Congo

red which

stains

the

cytoplasm.

The

FCR

stained

cells were less intensively stained than

_the

Giemsa stained cells. Therefore,

the

FCR

stained

micronuclei

were less

well

pronoun c.d.25

Consequent-ly, it

is

difficult

to score

manually, though

the

result is

reliable.

THE

MICRONUCLEI

ENUMERATION

METBOD

Manual enumeration

The proportion

of

micronucleated

cells

is

relativety

low,

consequently,

a lot

of

cells

have

to

be screened

(500-2000

cells).

Screening

a

large number

of

cells

manually

is

annoying

and takes a

lot of

time.

Besides,

staining using fluorescent

stains

which is more

reli-able, should pose

a

problem

in

manual enumeration,

due

to

the

limited

fluorescent time.

Flow-cytometric

enu

meration

Theoretically, Flow-cytometric enumeration can

be

done

on

fluorescent stained

cells.

A

flow-cytometric

enumeration

to

determine the frequency

of

micro-nucleated

polychromatic

erythrocytes

was done

using

Giemsa Giemsa

Feulgen-Congo red

Feulgen-Congored

MMS = methyl methane sulphonate,

*

resumed from Castelain

et a1.25

The

staining

methods

which

can

differentiate DNA

from other

substances

To

increase

the efficiency

of

the

micronucleus

test,

screening

in

each age

population

of

erythrocytes

is

needed.'For this

reason,

staining

methods

which

can

differentiate

DNA from RNA

are used; these methods

stain e.g. the acridine

o.ung",7

onin

Y,' and Hoechst

33342-addition,

the Feulgen-based

stain-ing

methods can also be used, e.g. the

Feulgen-Congo

red

(FCR) staining method which

was

applied

to

stain

the

binucleated lymphocytes.zs

The

staining

methods using

fluorescent stain

The acridine

orange

staining

is performed

by

immer-sion

of

slides

for

5 min in pH

7.4 sodium

phosphate

buffer

(l%

by weight)

containing 0.02 mg

of

acridine

orange

per ml, followed by a

10

min

rinse

in pH

7.4

phosphate

buffer. Slides are then wet

mounted

_and

examined

by

fluorescent microscopy using

a

Zeiss [image:5.595.43.280.313.407.2]

(6)

condi-Hoechst

33342-thiazole orange

stain.24

It

is

claimed

that

up to

70,000

polychromatic

erythrocytes can

be analyzeÀ

in

less

than

l0

minutes;

this

corresponds to

150-3,000

micronucleated polychromatic

erythro-cytes, 90-95%

of which

are

true

events as

determined

with

a

fluorescence microscope after

sorting.24

Automated enumeration using computer

based

Image analysis

The

automated enumeration

system

for

micronuclei

scoring consisted

of

a

Magiscan

MD

image processor

interfaced

to a

microscope

with

a

black/white

6-bit

TV

camera

with

Chalnicon

tube

and connected

to

a

motorized eight-slide

stage.

Automatic scoring

com-bined

with visual

inspection

was compared

to

manual

scoring

in

Giemsa

and Feulgen-Congo red

stained

cells. The result revealed

that

59-86%

of all

automat-ically

clasified

binucleated

cytokinesis-blocked

cells

were

correctly classified. Although

some

micro-nucleus were

overlooked during

automated

scoring,

on

average,

similar

micronuclei frequencies were

ob-tained

with

automated

and

-urrrul

s"oring.2s

U.ing

this automatic

system,

it

is

possible

to

scan

a

high

number of cells overnight, This

will

be

particularly

usefull in

scanning

tbe

feulgen-based

stained

cells,

in

which

the

micronuclei

are

difficult

to detect

manually,

due to

their

pale appearence.

CONCLUSION

The micronucleus test

is

a rapid

method

to

test

the

mutagenicity

of

various

agents.

Micronuclei can

be

analyzed

in

various

cells, in

vivo

using various

in-dividuals (human, animals, and plants),

or

in

vitro

using cultured cells. The most frequently

screened

cells

in in vivo

micronucleus

test are the

erythrocytes,

and

in in vitro

micronucleus

test are the

lymphocytes.

The

use

of

fluorescent stain

in

micronucleus

test

gives

a

more reliable result, but

the use

is

limited,

due to the

limited

fluorescent time. In

the

future, this

prob-lem can be

overcome using

Flow-cytometric

enumera-tion.

The

Feulgen-based

staining

of micronuclei

gives

a

less

well

pronounced image, thus

difficult

to

score

manually,

though the result is

reliable.

In the

future this

problem

can

be

overcome using

automated

enumera-tion

method.

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